During infection, bacteriophage T4 transcriptionally regulates the expression of its genome, resulting in early, middle, and late products. Middle RNA arises both from the firing of new middle promoters and the elongation of upstream transcripts past transcription termination or processing sites. As a model for understanding how the phage regulates middle RNA, we are studying the expression of a cluster of T4 genes (51 to 31): X.1 (newly identified DNA nuclease), uvsX (recombination protein), 40 (stimulates head formation), and 41 (DNA replication protein, part of the primase-helicase). Previously, we have mapped major 51 ends of the X.1 to 41 RNA to 225 bases upstream of X.1 and 190 bases upstream of uvsx. The regions upstream of these ends match the consensus sequence derived for middle promoters dependent on the T4 phage factor motA. We have demonstrated that these ends are indeed dependent on mota by Sl and primer extension analyses of RNA isolated from a su+ or suhost after infection of the T4 motA am mutant, amG1. The motA gene has been cloned into a multicopy plasmid vector. The presence of the motA plasmid complements both T4 amG1 and T4 tsG1 (a temperature-sensitive motA mutant) for growth of the phage in a host that restricts T4 motA mutants (E. coli tabG). MotA protein has been expressed from a clone in which the mota gene is downstream of a T7 promoter. Fractions containing. the expressed 25 kDa protein bind an oligomer containing the motA-dependent uvsx promoter sequence, retarding it during electrophoresis on polyacrylamide gels Previously, we have identified an open reading frame immediately upstream of the uvsx gene in a genetically unmapped region of the T4 genome. We have cloned the X.1 gene downstream of the lambda promoter PL and partially purified the expressed 25 kDa protein. Fractions containing X.1 protein bind DNA and in the presence of Mg++, convert supercoiled plasmid to nicked circle and discrete, linear products. This cutting activity is stimulated by uvsX protein, suggesting that X.1 may be involved in a recombination/repair pathway with uvsx.